Activated polyers and conjugates thereof

ABSTRACT

Macromolecular species such as enzymes, proteins, drugs, and solid supports which contain reactive carbonyl groups or groups which are readily converted to reactive carbonyl groups are modified by reaction with a compound of the formula ##STR1## in which R 1  is H, C 1  -C 20  alkyl, phenyl, (C 1  -C 10  alkyl)-substituted phenyl, or phenyl-substituted C 1  -C 10  alkyl; R 2  is C 2  -C 5  alkylene or (C 1  -C 4  alkoxy)-substituted C 2  -C 5  alkylene; R 3  is ##STR2## R 4  is H or C 1  -C 4  alkyl; n is at least 5; and m is zero or 1.

This is a continuation of application Ser. No. 07/164,307, filed Mar. 4,1988, now abandoned.

BACKGROUND OF THE INVENTION

This invention relates to the modification of macromolecular speciessuch as proteins, injectable compounds in general and solid supports, byconjugation with polymers. This invention further relates to activatedpolymers for use in such conjugation.

Polymers, such as polyethylene glycol, polypropylene glycol and analogs,have been coupled to enzymes, drugs, polypeptides and poly(amino acids)of various kinds to modify their immunological and biochemicalproperties. Among the desirable changes achieved in this manner are thereduction or elimination of immunogenicity, induction of tolerance, anincreased resistance to proteolytic degradation in the serum, acontrolled or gradual release (in the case of a drug) arising from aslow rate of enzyme-induced dissociation from the polymer, an alterationin the tissue distribution (in the case of a drug, for instance) byvarying its water solubility or its molecular size, a change in surfacecharacteristics (in the case of a solid support), and, in the case of anenzyme, for instance, a change in kinetics, a shift in the pH andtemperature optima, and altered reactivity to substrate.

The goal of conjugation in most cases is to modify certain propertieswhile retaining others (such as drug or enzyme activity, for instance)substantially unchanged. In many cases, this selectivity varies with thelocus of the conjugation site on the macromolecule, and the number ofsites on a single macromolecule which take part in the conjugation. Mostconjugations to date have been made at amino moieties on drugs orenzymes. In many cases, this presents a problem due to the widedistribution of reactive amino groups on the molecule. The result isoften a lack of control over the degree or rate of conjugation, and asubstantial interference with the desired activity of the macromolecule.In an immunoglobulin, for instance, where the antigen-binding portionsbear a large number of amino groups, conjugation at these groups cancause interference with the immunoglobulin s ability to bind antigen.

SUMMARY OF THE INVENTION

Novel compositions and methods are provided herein for the modificationof macromolecular species by conjugation with polymers at reactivecarbonyl groups on the species. Derivatized polymers for use inaccordance with the invention are those of the formula ##STR3## in whichR¹ is H or a protecting group which will prevent a coupling reactionfrom occurring at the end of the polymer to which it is attachedexamples of such protecting groups are C₁ -C₂₀ alkyl, phenyl, (C₁ -C₁₀alkyl)-substituted phenyl, or phenyl-substituted C₁ -C₁₀ alkyl;

R² is C₂ -C₅ alkylene or (C₁ -C₄ alkoxy)-substituted C₂ -C₅ alkylene;

R³ is ##STR4## R⁴ is H or C₁ -C₄ alkyl; n is at least 5; and

m is zero or 1.

Further included within the scope of this disclosure are analogs andhomologs of the above, varying for example in the identity and positionof the substituents and in other ways readily occurring to those skilledin the art, which may be considered as equivalents of the above.

Throughout this specification, the terms "alkyl," "alkoxy," "alkylene"and the like denote both straight-chain and branched-chain structures.

Conjugates formed as the reaction product of the macromolecular specieswith these derivatized polymers are those having the formula ##STR5## inwhich R¹, R², R³, R⁴, n and m are as defined above, R⁵ is either --N═ or--NH--, and A is the residue of the macromolecular species. The term"residue" is used herein in its conventional sense, meaning that portionof the molecule remaining after loss of the atoms removed from it in theprocess of conjugation. The residue may also vary with the type oflinkage as represented by the R⁵ group. Likewise, the choice between thetwo alternatives for R⁵ will depend in part upon the reactive group onthe macromolecular species and its neighboring atoms, and in part uponthe conjugation reaction. The various possibilities and the appropriateselection among them in any given case will be readily apparent to thoseskilled in the art.

The macromolecular species will be one bearing a carbohydrate orcarboxyl group available for conjugation. Carbohydrate groups are firstconverted to aldehydes to prepare them for the reaction. This is done byconventional means well known to those skilled in the art. Examplesinclude the use of galactose oxidase or periodate under mild conditions.

Within the scope of these formulas, certain embodiments are preferred,For example, R¹ is preferably H or C₁ -C₁₀ alkyl, with C₁ -C₅ alkyl morepreferred and methyl particularly preferred. Likewise, R² is preferablyC₂ -C₅ alkylene, with --CH₂ --CH₂ -- (ethylene) and --CH(CH₃)--CH₂ --(isopropylene) more preferred, and ethylene particularly preferred,Similarly, R⁴ is preferably H or methyl, with H particularly preferred.In further preferred embodiments, the group ##STR6## occupies either ameta- or para- position with respect to R³ on the phenyl ring. Theinteger n is preferably 10 to 1000, and most preferably 50 to 200.

The following are examples of activated polymers within the scope ofFormula I:

ω-Methoxy-α-(p-aminobenzoyl)polyethylene glycol

ω-Methoxy-α-(m-aminobenzoyl)polyethylene glycol [alternativenomenclature: poly(oxy-1,2-ethanediyl), α-(4-aminobenzoyl)-ω-methoxy]

ω-Methoxy-α-(p-aminobenzamido)polyethylene glycol

ω-Methoxy-α-(o-aminobenzamido)polyethylene glycol

ω-Methoxy-α-(p-aminophenylaminocarbonyl)polyethylene glycol

ω-Methoxy-α-(m-aminophenylaminocarbonyl)polyethylene glycol

ω-Methoxy-α-(p-aminophenylaminocarbonylamino)polyethylene glycol

ω-Methoxy-α-(o-aminophenylaminocarbonylamino)polyethylene glycol

ω-Methoxy-α-(p-hydrazinocarbonyl)benzamidopolyethylene glycol

ω-Methoxy-α-(p-hydrazinocarbonyl)benzoyl polyethylene glycol

ω-Methoxy-α-(m-hydrazinocarbonyl)benzoyl polyethylene glycol

ω-Ethoxy-α-(p-aminobenzoyl)polyethylene glycol

ω-Propoxy-α-(m-aminobenzoyl)polyethylene glycol

ω-Phenoxy-α-(p-aminobenzamido)polyethylene glycol

ω-n-Dodecyloxy-α-(m-aminophenylaminocarbonyl)polyethylene glycol

ω-n-Octadecyloxy-α-(p-aminophenylaminocarbonylamino)polyethylene glycol

ω-p-(n-Octyl)phenoxy-α-(p-aminobenzoyl)polyethylene glycol

ω-p-(n-Nonyl)phenoxy-α-(p-aminobenzoyl)polyethylene glycol

DETAILED DESCRIPTION OF THE INVENTION

The compounds of the present invention are prepared by conventionaltechniques well known among those skilled in the art, selected inaccordance with the desired substituent groups as listed above.

Compounds where R³ terminates in a carbonyl group at the end adjacent tothe phenyl group may be prepared by reacting a protected polyalkyleneglycol with an appropriately substituted benzoyl halide. To achieve the--NH--C(O)-- linkage, the protected polyalkylene glycol will besubstituted with an amino group at the α-position. To achieve the--O--C(O)-- linkage, the protected polyalkylene glycol can be useddirectly.

Compounds where R³ terminates in an amino group at the end adjacent tothe phenyl group may be prepared by reacting a protected polyalkyleneglycol with an appropriately substituted phenylisocyanate. To achievethe --NH--C(O)--NH-- linkage, an α-amino-substituted protectedpolyalkylene glycol is reacted with the phenyl isocyanate. To achievethe --O--CH₂ -- linkage, the protected polyalkylene glycol is reactedwith the appropriately substituted benzyl halide.

Attachment of the protecting group may be achieved in any conventionalmanner as well. A number of suitable mono-protected polyalkylene glycolsare commercially available and others are readily prepared by thoseskilled in the art.

The macromolecular species to which the activated polymers attach may beany of a wide variety of species containing a carbonyl group availablefor reaction with an amine, or species containing groups which arereadily convertible to reactive carbonyl groups, such as carbohydratemoieties. The macromolecular species may be enzymes, coenzymes,immunoglobulins, immunotoxins and other conjugates, proteins and drugsin general, and polymeric solid supports such as microbeads andchromatographic support media. Enzymes and coenzymes are of particularinterest. Examples are L-asparaginase, uricase, and catalases. Thecoupling reaction itself may be effected in accordance with conventionalprocedures for Schiff base reactions or amide bond formation, well knownto those skilled in the art.

The following examples are offered for purposes of illustration, and areintended neither to define nor limit the invention in any manner. Theseexamples illustrate the preparation of compounds within the scope of thepresent invention. In these examples, the following abbreviations areused:

IR: infrared spectra; values given in cm⁻¹ ; "s"=strong, "vs"=verystrong, "w"=weak, "sh"=shoulder

TLC: thin-layer chromatography: values given in R_(f) (ratio to thefront)

UV/VIS: ultraviolet/visible spectroscopy range; values given in relativeabsorption

EXAMPLE 1

A. Preparation of ω-Methoxy-α-(p-nitrobenzoyloxy)polyethylene Glycol.

This compound was prepared as a precursor toω-methoxy-α-(p-aminobenzoyl)polyethylene glycol, whose preparation isdescribed in part B which follows.

ω-Methoxy-α-hydroxypolyethylene glycol with an average molecular weightof approximately 5000 (0.90 g, 0.18 mMol) and p-nitrobenzoyl chloride(34 mg, 0.18 mMol) were refluxed in 25 mL of dry methylene chloride for5.5 hours, then stirred for two and one-half days at room temperature,Solvent was then removed in vacuo and the residue dried in vacuo to givea white solid weighing 0.85 g. IR analysis yielded the following:

IR: 1722 (C═O), 1530 (NO₂)

B. Preparation of ω-Methoxy-α-(p-aminobenzoyl polyethylene Glycol.

The product of part A of this Example (0.85 g) was dissolved in warmmethanol (100 mL) and treated with 10% Pd/C (200 mg, caution: sparks)and hydrogenated at 1 atmosphere for three days. The catalyst wasfiltered off and the solvent removed in vacuo to give a white solidweighing 0.72 g. The structure was confirmed as that ofω-methoxy-α-(p-aminobenzoyloxy)polyethylene glycol by TLC and IR asfollows:

TLC (90/10 CH₂ Cl₂ /CH₃ OH): one rather streaky spot at R_(f) 0.22-0.37(I₂ visible, not strongly UV positive); ninhydrin positive (startingmaterial is ninhydrin negative)

IR: 1695 (C═O), 1600, no absorbance at 1530 cm⁻¹

This material (55 mg) was dialyzed against H₂ O (3500 molecular weightcutoff) for 16 days at 4° C., then lyophilized. The UV/VIS spectrum andIR were basically unchanged as a result.

EXAMPLE 2

A. Preparation of ω-Methoxy-α-(m-nitrobenzamido)polyethylene Glycol.

This compound was prepared as a precursor toω-methoxy-α-(m-aminobenzamido)polyethylene glycol, whose preparation isdescribed in part B which follows.

ω-Methoxy-α-aminopolyethylene glycol with an average molecular weight ofapproximately 5000 (5.0 g, 1 mMol) in 75 mL of dry toluene was treatedwith 2.0 g (10.7 mMol) of m-nitrobenzoyl chloride at 80° C. in aconstant temperature bath under a nitrogen atmosphere for 18 hours. Thereaction mixture was concentrated to dryness in vacuo, then dissolved inH₂ O and dialyzed against H₂ O using 3500 molecular weight cutoffdialysis tubing for five days. A small amount of crude material whichdid not dissolve well in H₂ O remained at all times. The aqueoussolution was concentrated to dryness in vacuo at 50° C. and dried invacuo at room temperature to give a beige-colored solid weighing 2.4 g.Its structure was verified as that of the title compound by IR, UV/VISand TLC as follows:

IR (KBr): 2890 (s), 1730 (C═O), 1540 (NO₂), 1475, 1370, 1350, 1285,1250, 1120 (vs), 975, 850

UV/VIS: (CH₃ OH) 254 (0.18), 218 (0.48); (CH₂ Cl₂) 254 (0.96), 220(0.12)

B. Preparation of ω-Methoxy-α-(m-aminobenzamido)polyethylene Glycol.

The product of part A of this Example (2.4 g) was dissolved in 100 mL ofwarm ethanol, cooled, treated with 500 mg of 10% Pd/C and hydrogenatedat 20 psi on a Parr apparatus at room temperature for 16.5 hours. (Thewarming is necessary, with cooling before adding the catalyst. Onoccasion, the starting material will crystallize upon cooling.) Thecatalyst was filtered off and the filtrate evaporated in vacuo to give awhite solid weighing 1.98 g (82% yield). The structure was confirmed asthat of the title compound by IR, UV/VIS and TLC as follows:

IR (KBr): 2880 (s), 1720 (w, C═O), 1470, 1365, 1345, 1285, 1115 (vs),970, 845 (note that the peak at 1540 is no longer present)

UV/VIS: (CH₃ OH) 316 (0.068), 224 (0.815); (CH₂ Cl₂) 314 (0.247), 238(1.09)

TLC (4/1 C₂ H₅ OH/25% concentrated aqueous NH₃): R_(f) 0.6-0.7, onespot, gives blue fluorescence (the nitro starting material does notfluoresce); the product gives a weak reaction with ninhydrin.

EXAMPLE 3

A. Preparation of ω-Methoxy-α-(p-nitrophenylaminocarbonyl)-polyethyleneGlycol.

This compound was prepared as a precursor toω-methoxy-α-(m-aminophenylaminocarbonyl)polyethylene glycol, whosepreparation is described in part B which follows.

ω-Methoxy-α-hydroxy polyethylene glycol with an average molecular weightof approximately 5000 (0.90 g, 0.018 mMol) was dissolved in 25 mL of dryCH₂ Cl₂ with two drops of triethylamine. p-Nitrophenylisocyanate (30 mg,0.18 mMol) was then added and the mixture refluxed overnight. After 26hours, the solvent was removed in vacuo to give a yellow waxy solidweighing 0.78 g, whose structure was verified as that of the titlecompound by IR as follows:

IR (KBr): 1730 (carbamate), 1600, 1510 (sh), plus the usual absorptionsassociated with polyethylene glycol (1468, 1360, 1245, 1280, 1240, 1110,965, 845). Absorption at 2270 was not observed, indicating that nounreacted p-nitrophenyl isocyanate remained.

B. Preparation of ω-Methoxy-α-(p-aminophenylaminocarbonyl)-polyethyleneGlycol.

The product of part A of this Example (0.78 g) was dissolved in 100 mLof a warm 1:1 mixture of methanol and tetrahydrofuran, and hydrogenatedat 1 atmosphere with 10% Pd/C for 65 hours. The catalyst was filteredoff and the solvent removed in vacuo to give a pale brown solid weighing0.72 g, whose structure was confirmed as that of the title compound byTLC and IR as follows:

TLC (90/10 CH₂ Cl₂ /CH₃ OH): R_(f) 0.4 (faint), 0.23-0.3 (major), 0.18(minor). All three spots were ninhydrin positive.

IR (KBr): 1720, 1525 (w) plus the usual absorptions associated withpolyethylene glycol (see part A) Note: (1) The peak at 1600 cm⁻¹ in thestarting material (1595 in p-nitrophenylisocyanate), assigned to NO₂ isgone; and (2) C═O shows a shift from 1730 to 1720 cm⁻¹ similar toexamples tabulated by D. Dolphin and A, Wiese, Tabulation of InfraredSpectral Data, Wiley & Sons (1977).

The foregoing is offered primarily for purposes of illustration. It willbe readily apparent to those skilled in the art that variations andmodifications of the structures and procedures described herein may bemade without departing from the spirit and scope of the invention.

What is claimed is:
 1. A compound having the formula ##STR7## in whichR¹ is a member selected from the group consisting of H, C₁ -C₂₀ alkyl,phenyl, (C₁ -C₁₀ alkyl)-substituted phenyl, or phenyl-substituted C₁-C₁₀ alkyl;R² is a member selected from the group consisting of C₂ -C₅alkylene and C₁ -C₄ alkoxy-substituted C₂ -C₅ alkylene; R³ is a memberselected from the group consisting of ##STR8## R⁴ is a member selectedfrom the group consisting of H and C₁ -C₄ alkyl; n is at least 5; andthe amino group occupies either a meta- or a para-position with respectto R³ on the phenyl ring.
 2. A compound in accordance with claim 1 inwhich R¹ is a member selected from the group consisting of H and C₁ -C₁₀alkyl.
 3. A compound in accordance with claim 1 in which R¹ is a memberselected from the group consisting of H and C₁ -C₅ alkyl.
 4. A compoundin accordance with claim 1 in which R¹ is methyl.
 5. A compound inaccordance with claim 1 in which R² is C₂ -C₅ alkylene.
 6. A compound inaccordance with claim 1 in which R² is a member selected from the groupconsisting of --CH₂ --CH₂ -- and --CH(CH)₃ --CH₂ --.
 7. A compound inaccordance with claim 1 in which R² is --CH₂ --CH₂ --.
 8. A compound inaccordance with claim 1 in which R⁴ is a member selected from the groupconsisting of H and methyl.
 9. A compound in accordance with claim 1 inwhich R⁴ is H.
 10. A compound in accordance with claim 1 in which n is10 to
 1000. 11. A compound in accordance with claim 1 in which n is 50to 200.